Central nervous system (CNS) control of reproduction is mediated by a population of neuroendocrine cells that secrete gonadotropin-releasing hormone (GnRH). These GnRH neurons originate outside the CNS and migrate into the ventral telencephalon and hypothalamus during embryogenesis. Incorrect migration of GnRH neurons underlies some forms of reproductive disease, namely Kallmann Syndrome. However, the embryonic mechanisms regulating GnRH neuron migration and function are poorly understood. In part, this is because GnRH neurons are few in number and inaccessible in most vertebrate embryos or the intact adult brain. To overcome these barriers, we exploit several experimental features of zebrafish to identify GnRH neurons during embryonic migration and monitor electrophysiological activity in an intact neural network. The goal of this proposal is to elucidate the in vivo mechanisms of GnRH neuron migration and function. In transgenic zebrafish embryos, GnRH neurons express green fluorescent protein soon after birth and increase the rate of spontaneous action potential firing during embryonic migration. We hypothesize that increased electrical activity in GnRH neurons is an in vivo mechanism to regulate migratory rate, route, or destination. We will: 1) Determine when and where migratory GnRH neurons acquire neurophysiological activity, 2) Determine whether stereotyped migration is required for normal GnRH neurophysiology, and 3) Determine whether neuronal activity regulates embryonic migration of GnRH neurons. Experiments outlined in this proposal combine transgenic zebrafish, state-of-the-art microscopy, and single cell electrophysiology to monitor GnRH neuron migration and function in vivo. To determine the functional relationship between GnRH neuron migration and neuronal activity, we will disrupt GnRH neuron migration by manipulating mRNA expression of specific Kallmann genes. Conversely, we will use a GnRH neuron-specific transgene to overexpress a human inward rectifying potassium channel only in GnRH neurons and silence electrical activity during migration. Together, these analyses test the functional relationship between GnRH neuron migration and developmentally acquired electrical activity. Moreover, this system permits cell autonomous tests of human gene function (wild-type or mutant) using an in vivo model of GnRH neuron migration. We have developed a powerful model system and propose an innovative experimental approach to advance our understanding of GnRH neuron migration in vivo. A further understanding of the mechanisms guiding GnRH neuron migration is essential to determine the etiology of birth defects and predict clinical outcomes regarding reproductive maturation and fertility. These results in zebrafish complement ongoing clinical research efforts aimed toward identifying the function of (candidate) genes that underlie the molecular etiology of abnormal GnRH neuron migration in human reproductive disease. Public Health Relevance Statement: Several forms of human reproductive disease are caused by the inappropriate migration or function of specialized neuroendocrine cells that secrete gonadotropin-releasing hormone (GnRH). The goal of this proposal is to identify the molecular mechanisms and neurophysiology of embryonic GnRH neuron migration by exploiting the experimental features of transgenic zebrafish. A further understanding of the mechanisms guiding GnRH neuron migration is essential to determine the etiology of birth defects and predict clinical outcomes regarding reproductive maturation and fertility in humans.